The protein cytochrome c, Bcl-2 and Bax were assessed in the cytosolic and/or mitochondrial fractions of cortical neurons extracts in the presence/absence of AMD3100 treatment (200 nM, 24 h). Equal amounts of protein in a volume of 30 µL were separated on a 15% polyacrylamide gel (SDS-PAGE). These gels were transferred to a polyvinylidene fluoride (PVDF) membrane (GE Healthcare, Madrid, Spain). All membranes were blocked by adding 5% nonfat dry milk for 1 h at room temperature. The membranes were incubated overnight at 4 °C with the appropriate primary antibodies: goat polyclonal anti-cytochrome c for immunodetection (1/500) (sc-7159, Santa Cruz Biotechnology, Quimigen, Madrid, Spain), rabbit polyclonal anti-Bax (1/250) (sc-493), and anti-Bcl-2 (1/250) (sc-492) (Santa Cruz Biotechnology, Quimigen, Madrid, Spain) followed by incubation with peroxide-conjugated secondary antibodies for 1 h at room temperature (Santa Cruz Biotechnology, Quimigen, Madrid, Spain). Blots were developed by enhanced chemiluminescence (ECL select; GE Healthcare, Madrid, Spain) following manufacturer’s instructions. Anti β-actin (sc-47778) and anti-Tom-20 (sc-11415) antibodies (Santa Cruz Biotechnology, Quimigen, Madrid, Spain) were used as loading control for cytosolic and mitochondrial extracts, respectively.
Sc 7159
Manufactured by Santa Cruz Biotechnology
Sourced in Spain, United States
Sc-7159 is a laboratory instrument designed for protein analysis. The core function of this product is to facilitate the detection and quantification of specific proteins within a sample.
Automatically generated - may contain errors
Lab products found in correlation
Anti tom20 sc 11415, by Santa Cruz Biotechnology (1 mentions)
Peroxide conjugated secondary antibodies, by Santa Cruz Biotechnology (1 mentions)
Ecl select, by GE Healthcare (1 mentions)
Pvdf membrane, by GE Healthcare (1 mentions)
Anti β actin sc 47778, by Santa Cruz Biotechnology (1 mentions)
Ab15895, by Abcam (1 mentions)
Ecl plus kit, by GE Healthcare (1 mentions)
Sc 9416, by Santa Cruz Biotechnology (1 mentions)
Sc 9300, by Santa Cruz Biotechnology (1 mentions)
T9026, by Merck Group (1 mentions)
Ab31276, by Santa Cruz Biotechnology (1 mentions)
β actin, by Merck Group (1 mentions)
4 protocols using sc 7159
1
Cytochrome c, Bcl-2 and Bax Analysis
2
Western Blot Analysis of Apoptotic Proteins
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Total protein was extracted from cells using cell lysis buffer (0.5% NP-40, 0.5% SDS, 1.5 mM pH 7.4 Tris-HCL, and 15 mM NaCl). Protein samples (20 μg/lane) were electrophoresed, transferred to polyvinylidene difluoride (PVDF) membranes and incubated overnight with primary antibodies against cytochrome c (sc-7159, Santa Cruz Biotechnology, USA), Smac (sc-22766, Santa Cruz Biotechnology, USA), AIF (sc-9416, Santa Cruz Biotechnology, USA) and the mitochondrial loading control protein VDAC1/Porin (ab15895, Abcam). The membranes were treated with goat-anti-rabbit HRP-conjugated50 (link) secondary antibodies (Invitrogen Thermo Fisher, Waltham, Massachusetts, USA). The target protein bands were determined using the reagents visualized provided in the ECL +plus kit (GE Healthcare, Piscataway, NJ, USA) and the immunoreactive band intensities were analyzed with ImageJ (National Institutes of Health, Bethesda, MD, USA). The protein expression was quantified as optic density (OD) per mm2.
3
Protein Extraction and Immunoblotting for Muscle Samples
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Protein extracts from human quadriceps biopsies (20 μg) and from cells (40 μg) were obtained as previously described [23 (link),37 (link)]. Briefly, muscle powder from muscle biopsy specimens was homogenized in 1 ml buffer A [20 mM hydroxyethylpiperazine ethanesulfonic acid pH 7.5, 1 mM EDTA, 1 mM dithiothreitol, 1 mM MgCl2, 400 mM NaCl, 20% (v/v) glycerol, 0.75 mM spermidine, 0.15 mM spermine, 0.1% (v/v) Nonidet P40] with protease inhibitors. Primary muscle cells were lysed in hypertonic buffer containing 50 mM Tris pH7, 50 mM NaCl, 0.1% NP40, anti-proteases and 1 mM DTT. Samples were separated by SDS-PAGE and immunoblotted with an anti-ANT1 antibody (sc-9300, 1:1000, Santa Cruz Biotechnology, Le Perray-en-Yvelines, France), as previously reported [23 (link)]. Densitometry analyses were performed using Image J (NIH, Bethesda, MD) and values were normalized to loading controls (cytochrome c (muscle biopsies, sc-7159, 1:500, Santa Cruz Biotechnology), tubulin (primary muscle cells, T9026, 1:10000, Sigma Aldrich)). Protein extraction and western blotting were performed in X. laevis as previously described [45 (link)]. Vitellogenin was used as loading control.
4
Isolation and Characterization of Small EVs from MIA PaCa-2 Cells
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MIA PaCa-2 cells were cultured in in RPMI medium supplemented with EVs-depleted FBS for 72 hours in the presence of DMSO (5%) or Nexinhib20 (1 µM). Afterwards, EVs were isolated according to Crescitelli et al.76 (link)
Briefly, cells debris were eliminated through a centrifugation at 300 g for 10 min and a centrifugation at 2000 g for 20 min. Afterwards, supernatants were centrifuged at 16 500 g for 20 min to remove large vesicles and followed by 118 000 g centrifugation for 2.5 hour to collect small vesicles. All centrifugations were performed at 4°C.
For western blot, 20 µL of EV preparation from each condition was loaded into the gel. CD63 (1:500, BD Biosciences Cat# 556019, RRID:AB_396297) and β-actin (Sigma-Aldrich Cat# A3854, RRID:AB_262011) were used to detect EVs. Acetyl–cholinesterase (1:500, Abcam Cat# ab31276, RRID:AB_722529) and cytochrome C (1:200, Santa Cruz Biotechnology Cat# sc-7159, RRID:AB_2090474) were used to confirm purity of the small EVs isolated.
Briefly, cells debris were eliminated through a centrifugation at 300 g for 10 min and a centrifugation at 2000 g for 20 min. Afterwards, supernatants were centrifuged at 16 500 g for 20 min to remove large vesicles and followed by 118 000 g centrifugation for 2.5 hour to collect small vesicles. All centrifugations were performed at 4°C.
For western blot, 20 µL of EV preparation from each condition was loaded into the gel. CD63 (1:500, BD Biosciences Cat# 556019, RRID:AB_396297) and β-actin (Sigma-Aldrich Cat# A3854, RRID:AB_262011) were used to detect EVs. Acetyl–cholinesterase (1:500, Abcam Cat# ab31276, RRID:AB_722529) and cytochrome C (1:200, Santa Cruz Biotechnology Cat# sc-7159, RRID:AB_2090474) were used to confirm purity of the small EVs isolated.
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